KpC genes ESBL-genotype AmpC gene(s)
Species Strain CTX-M SHV SNP TEM SNP
E. coli DH5a n.d. n.d. n.d. n.d. n.d.
901200230 n.d. CTX-M Group 1 n.d. 238S n.d.
901200284 n.d. n.d. n.d. 104K + 238S DHA
901200288 n.d. n.d. n.d. n.d. ACT / MIR
901200370 n.d. CTX-M Group 1 n.d. 238S n.d.
100329-02 n.d. CTX-M Group 1 n.d. n.d. n.d.
100329-03 n.d. CTX-M Group 9 n.d. Wildtype n.d.
Klebsiella 32199 n.d. n.d. Wildtype n.d. n.d.
pneumoniae 6391 Kpc 2 n.d. Wildtype n.d. n.d.
6737 Kpc 3 n.d. Wildtype n.d. n.d.
n.d. = not detected
Genotyping of ESBL-, AmpC- and KPC-Genes
The genotyping of ESBL-genes was performed using the Check-MDR CT101 microarray
(Check-Points Health BV, Wageningen, The Netherlands) according to the manufacturer’s
instructions. In brief, whole-cell DNAs were extracted from overnight bacterial cultures using
QiaAmp DNA minikit (Qiagen, Hilden, Germany). Templates of the target β-lactamase DNA
sequences were generated during a multiplex ligation detection reaction step. These
templates are then amplified, and the products are hybridized in specific array tubes. The
tubes are then inserted in the single-channel ATR03 array tube reader upon completion of
the detection reaction, and images are acquired and interpreted with the software supplied
by the manufacturer (Check-Points). This software automatically translates the Check-MDR
CT101 microarray data into the presence or absence of a specific β-lactamase gene.
The kpc genes were PCR amplified using the primers KPC-42.for (5’-atg gcc gct ggc tgg ctt
tt-3’) and KPC-855.rev (5’-gag cgc agt cta gcc gca g-3’) leading to a 814-bp PCR
amplification product. Two microliters of total DNA was subjected to multiplex PCR in a 50-μL
reaction mixture. The mix contains 1× PCR buffer (10 mmol/L Tris–HCl [pH 8.3], 50 mmol/L
KCl), 1.5 mmol/L of MgCl2, 0.125 mmol/L of each deoxynucleotide triphosphate, 10 μmol/L
of each primer, and 2 U of AmpliTaq Gold Polymerase (Roche, Penzberg, Germany).
Amplifications were carried out with the following PCR conditions: 10 min at 94°C and 36
cycles of amplification consisting of 30 s at 94°C, 40 s at 52°C, and 50 s at 72°C, with 5 min
at 72°C for the final extension. DNA fragments were analyzed by electrophoresis in a 2%
agarose gel at 100 V for 1 h in 1× TAE (40 mmol/L Tris–HCl [pH 8.3], 2 mmol/L acetate, 1
mmol/L EDTA) containing 0.05 mg/L ethidium bromide. Sequencing of the kpc genes were
carried out with purified PCR amplification products (Qiatip)
The nucleotide sequences of both strands of the amplified DNA fragment were determined
using the same primers (KPC-42.for and KPC-855.rev ). Sequencing was performed at
Eurofins MWG Operon (Ebersberg, Germany) using an ABI Prism 1.1 Big Dye sequencing
kit and an ABI 3730 DNA analyzer (Applied Biosystems, Foster City, CA). Nucleotide
sequence analysis was performed using the Lasergene 8.0 software package (DNASTAR,
Madison, WI). The results were compared against the GenBank
(http://www.ncbi.nlm.nih.gov/) database using the BLAST algorithms. KPC variants and their
SNPs were compared to the following table in order to identify the respective KPC type:
1 atgtcactgt atcgccgtct agttctgctg tcttgtctct catggccgct ggctggcttt
61 tctgccaccg cgctgaccaa cctcgtcgcg gaaccattcg ctaaactcga acaggacttt
121 ggcggctcca tcggtgtgta cgcgatggat accggctcag gcgcaactgt aagttaccgc
181 gctgaggagc gcttcccact gtgcagctca ttcaagggct ttcttgctgc cgctgtgctg
241 gctcgcagcc agcagcaggc cggcttgctg gacacaccca tccgttacgg caaaaatgcg
301 ctggttccgt ggtcacccat ctcggaaaaa tatctgacaa caggcatgac ggtggcggag
361 ctgtccgcgg ccgccgtgca atacagtgat aacgccgccg ccaatttgtt gctgaaggag
421 ttgggcggcc cggccgggct gacggccttc atgcgctcta tcggcgatac cacgttccgt
481 ctggaccgct gggagctgga gctgaactcc gccatcccag gcgatgcgcg cgatacctca
541 tcgccgcgcg ccgtgacgga aagcttacaa aaactgacac tgggctctgc actggctgcg
601 ccgcagcggc agcagtttgt tgattggcta aagggaaaca cgaccggcaa ccaccgcatc
661 cgcgcggcgg tgccggcaga ctgggcagtc ggagacaaaa ccggaacctg cggagtgtat
721 ggcacggcaa atgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggcc
781 gtctacaccc gggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcg
841 gctagactcg cgctcgaggg attgggcgtc aacgggcagt aa
KPC variants and their SNPs
KPC variant SNP-147 SNP-308 SNP-716 SNP-814 GenBank ID
KPC-2 G C T C AY034847
KPC-3 G C T T AF395881
KPC-4 G G G C FJ473382
KPC-5 G G T C EU400222
KPC-6 G C G C EU555534
KPC-7 A C T T EU729727
KPC-8 G C G T FJ234412
KPC-9 G C C T FJ624872
KPC-10 G G T T GQ140348